1. Field of the Invention
The invention relates to modified oligonucleotides that are useful for studies of gene expression and for the antisense therapeutic approach. In particular, the invention relates to the synthesis of such modified oligonucleotides containing alkylphosphonate internucleoside linkages.
2. Summary of the Related Art
Antisense oligonucleotides are becoming increasingly recognized as powerful tools for inhibiting specific gene expression. The potential for using oligonucleotides as inhibitors of specific gene expression in an antisense therapeutic approach was first suggested in three articles published in 1977 and 1978. Paterson et al., Proc. Natl. Acad. Sci. U.S.A. 74: 4370-4374 (1977) discloses that cell-free translation of mRNA can be inhibited by binding a complementary oligonucleotide to the mRNA. Zamecnik and Stephenson, Proc. Natl. Acad. Sci. U.S.A. 75: 280-284 and 285-288 (1978) disclose that a 13-mer synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome can inhibit RSV replication in infected cell cultures and can inhibit RSV-mediated transformation of primary chick fibroblasts into malignant sarcoma cells.
Since these early studies, the ability of antisense oligonucleotides to inhibit virus propagation has become firmly established. Temsamani and Agrawal, Antisense Oligonucleotides as Antiviral Agents, In Advances in Antiviral Drug Design (Ed. E. de Clercq), JAI Press, Vol. 2, pp. 1-39 (1995), reviews the use of antisense oligonucleotides as antiviral agents. More recently, antisense oligonucleotides have also been developed as anti-parasitic agents and have shown promise as candidates for therapeutic applications for diseases resulting from expression of cellular genes. The development of various antisense oligonucleotides as therapeutic and diagnostic agents has recently been reviewed by Agrawal and Iyer, Current Opinion in Biotechnology 6: 12-19 (1995).
As interest in the antisense therapeutic approach has grown, various efforts have been made to improve the pharmacologic properties of oligonucleotides by modifying the sugar-phosphate backbone. Agrawal and Iyer, Curr. Op. Biotech 6: 12-19 (1995) reviews the use of methylphosphonate linkages in oligonucleotides. Although oligonucleoside methylphosphonates have shown serious limitations as antisense agents, chimeric oligonucleotides containing methylphosphonate linkages have shown greater promise. U.S. Pat. No. 5,149,797 describes chimeric oligonucleotides having a phosphorothioate core region interposed between methylphosphonate regions.
Various methodologies have been used to synthesize oligonucleotides containing methylphosphonate internucleoside linkages. Miller et al., Biochemistry 25: 5092-5095 (1986), discloses an early methodology using a polymer support. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches a more generally applicable phosphoramidite approach using a controlled pore glass (CPG) support. All of the existing approaches, however, are inherently limited by the susceptibilty of the methylphosphonate linkage to hydrolysis by base, which precludes the use of the usual deprotection step, which employs prolonged treatment with 28% ammonium hydroxide. Some attempts to deal with this problem have included the use of N-isobutyryl-protected cytidine nucleoside phosphonamidite monomers in conjunction with dA.sup.bz and dG.sup.iBu monomers, followed by deprotection using initial exposure of the oligomer to 10% ammonium hydroxide in acetonitrile/ethanol at room temperature, then prolonged exposure to ethylenediamine. Also used has been pretreatment of the protected oligonucleotide with hydrazine hydrate in pyridine/acetic acid, followed by prolonged exposure to ethylene diamine/ethanol. Although these approaches have provided somewhat inconvenient answers to certain problems, they have created problems of their own for large scale synthesis of chimeric oligonucleotides, which have segments of different internucleosidic linkages. For example, the dG.sup.iBu -methylphosphonamidite monomer is insoluble in acetonitrile, which is the solvent commonly used with most other phosphoramidite monomers. Consequently, prior to each coupling step at which this monomer is added, it is necessary to thoroughly wash the monomer delivery lines and the synthesis column with a solvent in which the dG.sup.iBu monomer is soluble, such as anhydrous peroxide-free THF or CH.sub.3 CN/CH.sub.2 Cl.sub.2, to avoid precipitation of this monomer in the delivery lines or column. There is, therefore, a need for new synthesis processes which will provide convenient synthesis of chimeric oligonucleotides containing methylphosphonate internucleoside linkages in conjunction with other types of internucleoside linkages. Ideally, such a process should provide a convenient single step deprotection, should allow all of the monomers to be delivered in the same solvent, and should be adaptable to different chemical synthesis approaches.